The biotechnology market is the fastest growing sector within the world pharmaceutical market accounting for 18% ($130 bn) of all market sales in 2010. This growth from 10% of the market share in 2002 is set to grow 48% between 2010-2016 from $130 bn to $192 bn [1]. Biopharmaceutical therapeutics encompasses four main biomolecule types: recombinant proteins, monoclonal antibodies (MAbs), viral vaccines, and plasmid DNA [2]. There are currently over 200 MAb products on the market with over 1000 in clinical trials [3]. As with most therapies there is a global pressure to continue developing new biotherapuetics while driving down the cost of production increasing their availability to a wider scope of the population [4,5]. This is particularly apparent to the downstream bioprocess (DSP) involved with the purification of biomolecules where chromatography alone accounts for over 50% of the cost of goods (COGs). Advancements in upstream processes over the last 15 years have seen fermentation titres grow from 0.5 g/L-50 g/L, and with this not being mimicked in the DSP there has been a clear drive in industry and academia for development [6,7].
Conventional chromatography involves various techniques for the separation of mixtures by passing a mobile phase through a stationary phase. The analyte (load/feed) is the mobile phase which is passed through the solid stationary phase in the form of a packed bed system based on adsorbent beads 50-100 μm in diameter. This stationary phase has differing affinities for different species contained in the load. The species that are either wanted (product capture) or not wanted (contaminant capture) bind to the stationary phase allowing for the purification of the product stream.
Affinity chromatography, developed by Pedro Cuatrecasas and Meir Wilchek [8], separates on the basis of a reversible interaction between a biomolecule and a specific ligand coupled to a chromatography matrix. The ligand most commonly used is Protein A due to its high selectivity which means equipment only needs to be scaled for product output requirements [9].
Other ligands in use include Protein G, Protein A/G and Protein L. Each has a different binding site recognizing different portions of antibodies which becomes useful when the product are antibody fragments as opposed to a whole MAbs [10,11].
Ion exchange chromatography is a widely used technique for the separation and purification of proteins, polypeptides, nucleic acids, due to its widespread applicability, high resolving power, high capacity and simplicity. Separation in ion exchange is obtained as biomolecules have ionisable chemical moieties which render them susceptible to charge enhancement as a function of ionic strength and pH. This implies biomolecules have differing degrees of interaction with an ion exchanger (insoluble matrix to which charged groups have been covalently bound) due to differences in their charges, charge densities and distribution of charge on their surface.
At a pH value below its PI (isoelectric point) a protein (+ve surface charge) will adsorb to a cation exchanger (−ve). At a pH above its PI a protein (−ve surface charge) will adsorb to an anion exchanger (+ve).
Consequently under a set of defined mobile phase conditions a biopolymer mixture may be chromatographed using an ion-exchange medium in suitable contactor. Dependent on the relative ionic charge of the components, some biopolymers will adsorb (adsorbates) and others will remain in solution. Desorption of bound material can then be effected resulting in a degree of purification of the target biomolecule [12].
The projected increase in the number of antibody therapies over the next 4 years along with improvements in upstream productivity, and process economics gives a requirement for improved downstream processing techniques. The limitations of chromatography systems are already being seen in the form of expensive resins and throughput volumes which gives strong argument to the demand for improving technologies in this field.
There are two major drawbacks of traditional packed bed column chromatography; pressure drop and residence times. The operational flow rates in a packed column are limited by the pressure drop across the column. The compressible packed bed is susceptible to high pressures which can have drastic consequences with implosion of the column itself. At such a stage in the bioprocess where material is extremely high value this can have severe economic impacts for any company. Packed bed columns usually employ porous ca. 50 μm diameter Agarose beads as highly porous structures that achieve a suitable surface area for adsorption. However the system relies on diffusion for the large target biomolecules to come into contact with these surfaces which requires long residence times. As such the flow rates must be kept at a relatively low value, often in the range of 100 cm/h, which therefore limits the throughput of the system. The concern here is regarding the inefficient use of expensive chromatography resins [13,14].
Membrane adsorbers have been commercially available for many years now, but these have only proven to be useful at small scale [15]. Using membrane chromatography allows for operation in convective mode thereby significantly reducing the diffusion and pressure drop limitations seen in column chromatography. Operating at much higher flow rates offers advantages such as decreasing process time thereby increasing throughput and reducing damage to product biomolecules due to shorter exposure to unfavourable medium [16].
Kalbfuss, et al. described how the use of a commercially available anion exchange membrane proved to be a potentially viable option for the removal of viral particles operating in contaminant capture mode with relatively high flow rates of 264 cm/h performing consistently well [21].
Zhou and Tressel carried out a cost analysis based on their experimentation using anion (Q) exchange membranes to remove four model viruses. Results suggested that the economic viability of the Q membrane over the Q column was highly dependent of production scale along and specific to each production process [22]. Research suggests that there is potential for Q membranes though the advantages do not seem significant enough for the industry to adopt such a significant change. Additionally, chromatographic operations that run in capture mode such as cation-exchange chromatography (CEX) and affinity chromatography have proven to be much more challenging with poor peak resolution observed [23]. One aspect working in the favour of membrane chromatography systems is the increased uptake of disposable systems in industry, of which membranes hold many advantages over conventional packed bed columns.
There are many examples of membrane adsorbers in use and in continuing development [24-29].
It is clear from the work that is already published on membrane adsorbers that several important properties are required relating specifically to the membrane structure. For efficient utilisation of binding surface area the inlet flow must have even dispersion and the pore size distribution must be small so as to minimize any channelling. Membranes used in the chromatography method discussed above also excel when combined with other technologies.
Simulated Moving Bed (SMB) technology has been in use for many years in the chemical industry, originally developed for difficult petrochemical separations [32]. Later its use in the pharmaceutical industry quickly grew due to its strong ability to perform chiral separations with the first US Food and Drug Administration's (FDA) approved drug manufactured by SMB technology reaching the market in 2002 with Lexapro [33]. Traditionally the powers of SMB to carry out separation based on the different moieties of complex components have been focused on systems that yielded poor productivity using column chromatography. Today however more focus is being placed on bind/elute chromatographic processes in an effort to improve the utilisation of expensive adsorptive resins and reduce the large volumes of buffers used at large scale production [34,35].
In this fashion SMB chromatography operates by employing three or more fixed adsorbent substrates, such as packed bed columns, with buffer and feed streams flowing into to system continuously. A counter-current solid substrate is simulated by switching various valve inlet ports periodically.